The E.Coli Heat Labile Enterotoxin B-Subunit Enhances CD8+ T-Cell Killing Of An Epithelial Tumour Cell Line Expressing EBV LMP2

EBV LMP2 is a potential tumour immunotherapeutic target but EBV lymphoblastoid cell lines (LCL) are not normally susceptible to killing by uncloned LMP2-specfic CD8+ cytotoxic T-cells (CTL) in vitro unless pulsed with epitope peptide or if LMP2 is expressed by vaccinia virus. However, the E. coli enterotoxin subunit, EtxB, alters the MHC class I-dependent processing of LMP2 and enhances susceptibility of LCL to CTL. We wished to determine if EtxB could enhance the LMP2-specific CTL susceptibility of an oropharyngeal tumour cell line artificially expressing LMP2. The HLA A*02 oropharyngeal carcinoma cell line, H103, was stably transfected with an expression vector containing the LMP2A coding sequence. These cells (H103 LMP2) were pre-incubated with EtxB or the HLA A*02- restricted LMP2 epitope, CLGGLLTMV (CLG), or were untreated. LMP2-specific CTLs were recovered and expanded from an HLA A*02 donor and added to the H103 LMP2 target cells at an E:T ratio of 12:1. The expression and distribution of LMP2 and EtxB in the H103 LMP2 target cells was assessed by Western blotting and confocal immunofluorescence microscopy. LMP2 protein expression was exclusively intracellular in H103 LMP2 epithelial cells and partially co-localized with internalized EtxB and a Golgi marker after 4 hours. Control H103 cells were only killed by LMP2-specific CTL when pretreated with CLG peptide. H103 LMP2 cells were also not killed unless pre-treated with CLG peptide. However, EtxB treatment of H103 LMP2 cells rendered them susceptible to killing by LMP2-specific CTL without pre-exposure to CLG. As with EBV LCL, the susceptibility to LMP2-specific CTLs of epithelial tumour cells expressing LMP2 protein was greatly enhanced by treatment with EtxB and that cell surface LMP2 expression is not a requirement for this effect to take place. These results further strengthen the view that EtxB has potential as a therapeutic agent for EBV-associated tumours expressing LMP2.